Charged pyridinium oximes with thiocarboxamide moiety are equally or less effective reactivators of organophosphate-inhibited cholinesterases compared to analogous carboxamides

Abstract The organophosphorus antidotes, so-called oximes, are able to restore the enzymatic function of acetylcholinesterase (AChE) or butyrylcholinesterase (BChE) via cleavage of organophosphate from the active site of the phosphylated enzyme. In this work, the charged pyridinium oximes containing thiocarboxamide moiety were designed, prepared and tested. Their stability and pKa properties were found to be analogous to parent carboxamides (K027, K048 and K203). The inhibitory ability of thiocarboxamides was found in low µM levels for AChE and high µM levels for BChE. Their reactivation properties were screened on human recombinant AChE and BChE inhibited by nerve agent surrogates and paraoxon. One thiocarboxamide was able to effectively restore function of NEMP- and NEDPA-AChE, whereas two thiocarboxamides were able to reactivate BChE inhibited by all tested organophosphates. These results were confirmed by reactivation kinetics, where thiocarboxamides were proved to be effective, but less potent reactivators if compared to carboxamides.


Introduction
Organophosphates (OP) widely used as pesticides (e.g. chlorpyriphos, parathion) are synthetically prepared compounds that can also be misused as chemical weapons (e.g. nerve agents sarin, soman, tabun, VX) 1 . They are stable, and have a rapid effect, primary in the nervous system, after being absorbed by the skin and respiratory system 2,3 .
OP are irreversible inhibitors of ChEs, acetylcholinesterase (AChE, EC 3.1.1.7) and butyrylcholinesterase (BChE, EC 3.1.1.8). While inhibition of AChE can lead to life threatening intoxication due to cholinergic overstimulation and crisis, inhibition of BChE has no direct adverse effects. For this reason, BChE can be used as the OP bioscavenger and it is considered as a pseudo-catalytic bioscavenger 4 . The mechanism of AChE inhibition implies rapid phosphylation of the Ser203 hydroxyl group in AChE. After that AChE is no longer able to hydrolyse acetylcholine (ACh) which leads to its accumulation in the postsynaptic cleft followed by the overstimulation of cholinergic receptors 5,6 . Symptoms of poisoning are e.g. sweating, diarrhoea, tremors and muscle spasms. Death may be caused by respiratory system failure 6 .
For this reason, the molecular design of charged pyridinium oximes with thiocarboxamide moiety was proposed from K027 (5), K048 (6) and K203 (7). The pyridinium scaffold with oxime moiety was retained as the a-nucleophile that is able to attack OP-inhibited cholinesterase. Similarly, the second aromatic moiety and the connecting linker remained to be same as in the parent molecules. Differently, the carboxamide moiety was replaced by thiocarboxamide to investigate the sulphur involvement in the binding of reactivator's molecule within the active site of AChE or BChE. The carboxamide fragment was formerly found to play an important role in hydrogen bonding of charged oximes in the peripheral site of AChE 22 . For this reason, the use of spatially bulkier thiocarboxamide moiety could change such hydrogen bonding and overall affinity towards particular cholinesterase or it could increase the affinity to OP-enzyme complex for better reactivation.
In this work, the novel bis-pyridinium oximes were designed, synthetised and evaluated. The stability, oximate formation properties, inhibition of human ChEs and reactivation of human OPinhibited ChEs was studied.

Chemistry
All chemicals used for the synthesis were purchased from Sigma-Aldrich (Prague, Czech Republic) in the highest available purity. All used solvents were supplied by Penta Chemicals Unlimited (Prague, Czech Republic). Organophosphorus compounds 4-nitrophenyl isopropyl methylphosphonate (NIMP, sarin surrogate), 4-nitrophenyl ethyl methylphosphonate (NEMP, VX surrogate), 4-nitrophenyl ethyl dimethylphosphoramidate (NEDPA, tabun surrogate) and paraoxon (POX) were purchased from Chemforase (Mont-Saint-Aignan, France). Thin layer chromatography was performed on Merck silica gel 60 F 254 and Merck cellulose F analytical plates. Detection was carried out with ultraviolet light (254 nm). Melting points were recorded on a Melting Point Apparatus -B€ uchi B-545 (Donau Lab, Czech Republic) without correction. The 1 H and 13 C NMR spectra were measured in DMSO-d 6 solution at room temperature on FT NMR spectrometer Avance NEO 500 MHz (499.87 MHz for 1 H and 125.71 MHz for 13 C) (Bruker, Germany). Chemical shifts, d, are given in parts per million (ppm) and spin multiplicities are given as br s (broad singlet), s (singlet), d (doublet) or m (multiplet). Coupling constants, J, are expressed in hertz (Hz). For 1 H d is relative to DMSO-d 6 (d ¼ 2.50) and for 13 C is relative to DMSO-d 6 (d ¼ 39.43). High Resolution Mass Spectrometry (HRMS) was determined by Q-Exactive Plus hybrid quadrupole-orbitrap spectrometer.
General procedure for synthesis of monoquaternary salts 8-10 The monoquaternary salts 8-9 were prepared by reaction of 4-hydroxymethylpyridine (32.8 mM) and dibromoalkane (163.8 mM) in acetone (30 ml). The mixture was stirred at reflux for 8 h, then cooled to room temperature and filtered and washed with acetone (3 Â 20 ml). The crude product was crystalised from acetonitrile (100 ml per 1 g of product) at reflux and filtered under reduced pressure. The filtrate was evaporated under the reduced pressure to produce monoquaternary salts 8-9 23,24 .
General procedure for synthesis of bisquaternary salts 11-13 Secondly, the synthesis of the bisquaternary salt was completed. To a solution of monoquaternary salt 8-10 (1.48 mM) in dimethylformamide (DMF, 1.40 ml) was added 4-pyridinethioamide (2.22 mM). The resulting mixture was stirred at 60 C for 48 h. The solvent was concentrated under the reduced pressure and the crude product was purified by crystallisation from acetonitrile at reflux. Then the solid was filtered, washed with acetonitrile and dried under the vacuum.

Stability determination
The stability of tested compounds was evaluated in two different mediademineralised water and phosphate buffered saline (PBS; Sigma-Aldrich P4417) as simulation of physiological environment. Compounds were dissolved in particular solvent (final concentration 1 mg/mL) and incubated at 37 C. The amounts of analysed compounds were determined at time intervals of 0, 1, 2, 3, 4 and 5 h by UHPLC Infinity II 1290 system (Agilent Technologies, Santa Clara, USA) coupled with DAD detector. The stability was evaluated as the amount of remaining compound (%) in sample after specific time interval. GraphPad Prism version 8.2 (San Diego, USA) was used for the statistical data evaluation and visualisation. pK a determination The negative decimal logarithms of the dissociation constants (pK a ) of prepared compounds were determined spectrophotometrically using buffers of given pH (range from 4.5 to 10.0 with 0.5 unit increment). Ten microliters of tested oxime (1 mg/mL) were dissolved in 490 mL of particular buffer and absorbance spectra of various dissociation states were scanned in range 200-400 nm using Carry-60 UV-VIS spectrophotometer (Agilent Technologies, Santa Clara, USA) at 20 C. The pK a values were calculated from the sigmoidal dependence of the absorbance of the dissociated form of the substance on the pH value using GraphPad Prism 8.2 software (San Diego, USA).

Inhibition assay
Recombinant forms of human acetylcholinesterase (hrAChE) and butyrylcholinesterase (hrBChE) were prepared at the Department of Chemistry, Faculty of Science, University of Hradec Kralove 26 . The inhibitory effect of tested oximes on hrAChE/hrBChE was determined by standard Ellman method adapted for 96-well plates 27 . The reaction mixture consists of hrAChE (final protein concentration 70 ng/mL) or hrBChE (220 ng/mL of protein), water solution of tested compound at appropriate concentration (the range of 1 mM to 500 mM for hrAChE and of 50 mM to 1500 mM for hrBChE) and solution of 5,5 0 -dithiobis-2-nitrobenzoic acid (DTNB, final concentration 500 mM, pH 7.4) in 20 mM Na-phosphate buffer. The mixture was pre-incubated for 15 min at 37 C. Afterwards the substrate acetylthiocholine iodide (ATCI) or butyrylthiocholine iodide (BTCI) was added to the final concentration of 1000 mM. The total volume of reaction was 100 mL. Creation of the product 5-thio-2-nitrobenzoic acid (TNB), formed during the reaction was determined by observing its absorbance at specific wavelength 436 nm. The catalytic activity of enzyme was evaluated as amount of product (%) formed in the reaction after 10 min of incubation at 37 C. IC 50 values of compounds were calculated using non-linear regression by GraphPad Prism 8.2 (San Diego, USA). Data were calculated from three individual experiments which were made in triplicate.

Reactivation screening
Recombinant enzymes (hrAChE/hrBChE) were inhibited by 25 mM POX, NEMP, NIMPor NEDPA for 30 min to obtain >99% inhibition. The excess of organophosphate was removed by dialysis against 25 mM Na-phosphate buffer (pH 7.4) for 16 h with three buffer exchanges. The tested oxime (10 mM or 100 mM) was incubated with inhibited enzyme for 15 or 30 min at 37 C. The reaction mixture (100 mL) contained 10 mL of inhibited enzyme (1.95 ng of total protein), 20 mL of DTNB (2.5 mM), 10 mL of relevant oxime solution and 50 mL of Na-phosphate buffer (25 mM, pH 7.4). In parallel, reaction mixture containing no enzyme was followed as blank reaction to reflect oximolysis. The reaction was started by addition of 10 mL of substrate ATCI or BTCI (10 mM). The catalytic activity of enzyme reactivated by oxime was determined spectrophotometrically at specific wavelength 436 nm using Spark multimode microplate reader Tecan (Mannedorf, Switzerland). Data were calculated from three individual experiments which were made in triplicate.

Reactivation kinetics
The selected compounds with promising reactivation ability were further tested to investigate reactivation kinetics parameters. Inhibited enzyme (hrAChE/hrBChE) was incubated for eight different times (0.5 to 15 min) with seven different concentrations of tested oxime (varying from 1 to 1200 mM) at 37 C. As the blank the mixture containing no enzyme (to control oximolysis) was used. Acquired data were analysed by non-linear regression analysis according to Worek et al. 28 using GraphPad Prism 8.2. Data were calculated from three individual experiments which were made in triplicate.

Chemical synthesis
The monoquaternary precursors 8-10 were formerly prepared by our research group [23][24][25] . Subsequent bimolecular nucleophilic substitution (S N 2) of these monoquaternary salts with 4-pyridinethioamide in aprotic polar solvent DMF allowed creation of bisquaternary salts 11-13 (Scheme 1). The yields of these reactions ranged from 36% to 61%. The lowest yield was obtained in case of compound 11 (K487) which was probably caused by steric hindrance of two close pyridinium rings. The highest yield was obtained for compound 13 (K489), which contains but-2-en-1,4diyl chain. In this case, the substrate 10 react rapidly by the S N 2 mechanism because the p-system of the adjacent double bond can stabilise the transition state by conjugation. The final compounds were determined by NMR (Supplementary Figures 1-6) and HRMS analysis. Based on NMR and HRMS analysis, the noncalibrated purity of all products was !95%.

Stability determination
Prepared compounds K487 (11), K488 (12) and K489 (13) and analogous oximes K027 (5), K048 (6) and K203 (7) were tested for stability in unionised water and PBS at 37 C for 5 h, when the buffered environment and selected temperature are better mimicking human relevant conditions and the selected time is close to a full elimination of similar oximes from human organism. All compounds showed high stability in both media after 5 h ( Figure  3). The degradation of tested oximes in pure water was found negligible. Some degradation of K489 (13) and K203 (7) was found in PBS buffer, but still with residual >90% oxime after 5 h. Stability is very important parameter for the compound use and also for maintaining the biological activity for the necessary time i.e. first 1-2 h after OP intoxication for charged oximes 29 .
The pK a values were determined for compounds K487 (11), K488 (12), K489 (13), pralidoxime (1), asoxime (4) and for parent oximes (5)(6)(7). The pK a is an important parameter in pharmaceutical development to rationalise the physicochemical and biopharmaceutical properties of the drug molecule. It allows to detect protonated/ deprotonated form of the compound under physiological conditions. In case of oximes deprotonated form so called oximate anion is important, because it is the active form of the oxime reactivator 30 . Change of amidic group into thioamidic group did not caused significant changes in the pK a values of oxime moiety (Table 1). This result was expected because sulphur does not differ much in electronegativity compared to oxygen. Moreover, thiocarboxamide moiety is far distanced to affect the electron density at the oxime pyridinium ring.
Taken together, pralidoxime (1) and asoxime (4) were found to be generally less potent reactivators of NEMP-, NEDPA-and POX-inhibited AChE, when compared to carboxamides (5-7), while thiocarboxamides (11)(12)(13) resulted as weaker reactivators if compared to carboxamides. This result is most probably related to the stronger inhibition of AChE provided by thiocarboxamides which decreases overall activity of reactivated enzyme.

Reactivation kinetics
For reactivation kinetics, only few thiocarboxamides with promising reactivation screening parameters were selected and their carboxamide analogues were chosen for comparison. Compound 11 was chosen for kinetic experiments in case of NEDPA-inhibited AChE, due to its highest reactivation ability in the screening and compounds 11-12 were selected in case of NIMP-inhibited BChE. The affinity of relevant oxime towards OP-inhibited recombinant enzyme (reflected by K D ) and the ability to remove the OP residue from the active site of the enzyme (reflected by k r ) were determined. Afterwards, the specific overall second-order reactivation rate constant (k r2 ) was calculated (k r2 ¼k r /K D ). The highest affinity for NEDPA-inhibited AChE, as well as the fastest dephosphorylation of catalytic serine (k r ) was exhibited by oxime K027 (5), which therefore resulted with the highest k r2 constant. Thiocarboxamide 11 was found to be a better reactivator of NEDPA-AChE than 2-PAM (1) and asoxime (4), but less effective than K027 (5) ( Table 2). In addition, the reactivation kinetics of 5 and 11 towards NEDPA-AChE was directly compared by using 50 mM concentration of the reactivator (Figure 7). In this case, oxime 11 showed markedly lower reactivation which is caused by its stronger inhibition of AChE (IC 50 $ 10 mM), which indicates more convenient use of carboxamide moiety instead of thiocarboxamide one when using the same (trimethylene) linker. NIMP-BChE conjugate showed the highest affinity for oxime K027 (5) and its analogue thiocarboxamide 11. Furthermore, due to the high reactivation rate (k r ) these oximes can be singled out as the best reactivators of NIMP-inhibited BChE with an overall reactivation rate of about 6-fold higher than 2-PAM and HI-6 ( Table 2). The reactivation kinetics of 5 and 11 towards NIMP-BChE was also directly compared by using 100 mM concentration of the reactivator (Figure 7). Oxime 11 resulted as slightly weaker reactivator to its carboxamide analogue 5 which is again related to its slightly stronger inhibition of BChE.
The change of carboxamide into thiocarboxamide moiety doesn't bring much benefit for reactivation of organophosphates and the presence of thiocarboxamide group causes unwanted higher inhibition of AChE/BChE and thus decreased reactivation.

Conclusion
Three pyridinium mono-oximes containing thiocarboxamide group were designed, synthetised and tested. The novel compounds were evaluated for stability, and all compounds resulted as highly stable in both media (water and PBS). Further, pK a was determined and compared to parent oximes or standard compounds and no significant changes in the pK a values were found. The inhibitory effect of tested compounds on hrAChE and hrBChE was tested, and the determined IC 50 s were in the low mM range for hrAChE unlike for hrBChE. The novel compounds were evaluated for reactivation of hrAChE and hrBChE inhibited by NIMP (sarinsurrogate), NEMP (VX-surrogate), NEDPA (tabun-surrogate) and paraoxon. From reactivation screening, compound K487 (11) showed ability to reactivate AChE inhibited by NEMP and NEDPA. For BChE screening, K487 (11) and K488 (12) were found to have ability to reactivate all tested OPs. The reactivation kinetics was further determined for thiocarboxamides K487 (11) and K488 (12). Oxime 11 resulted as slightly weaker reactivator of NEDPA-AChE or NIMP-BChE if compared to its direct carboxamide analogue 5, which is caused by its higher intrinsic inhibitory ability for both enzymes. Taken together, the tested thiocarboxamides resulted as equally or less effective charged oxime reactivators when compared to their carboxamide analogues. Therefore, the introduction of thiocarboxamide moiety doesn't bring much benefit to the overall cholinesterase reactivation.